Collagen is a protein comprising the major fibrous element of mammalian skin, bone, tendon, cartilage, blood vessels, and teeth. Its biological purpose is to hold cells together in discrete units; and secondarily it has a directive role in developing tissues. In mammals, collagen is the principal protein and comprises ten percent of the total protein content of the body.
The collagen proteins are distinctive in their physical characteristics in that they form insoluble fibers possessing high tensile strength. It is the fibrous nature of the collagen that serves to hold the various body structures and components together. In fact, the word "collagen" is derived from the Greek words meaning "to produce glue". Thus collagen is a vitally important biological protein.
While the molecular structure is modified to meet the needs of particular tissues, all collagens are organized into a common structure consisting of three polypeptide chains that form a triple stranded helix. These triple stranded helical units, in turn, are formed into a quarter-staggered array of linearly aligned bundles which make up collagen fibers. The collagen fibers are stabilized by covalent cross-links. Two kinds of cross links are formed, those which are intramolecular between the helically stranded polypeptide chains; and those which are intermolecular between different helical units.
These cross-links principally occur in the non-helical ends of the peptide chains wherever lysine, or hydroxylysine amino acid residues occur. Such cross-links are generated between the lysine or hydroxylysine residues through either an aldol condensation or Schiff base reaction. In the first type of reaction the .epsilon.-amino group of a lysyl residue is converted into aldehyde by the enzyme, lysyl oxidase. If sufficiently adjacent one another, two such aldehydes undergo an aldol condensation to form an aldol condensation product. In the second type of reaction, aldehydes derived from lysyl or hydroxylysyl groups can also condense with the .epsilon.-aminogroup of lysyl or hydroylysyl residues to form Shiff base cross-links.
In some instances the aldol-lysine cross-links can react with a histidine side chain to form an histidine-aldol cross-link. In other instances, the aldehyde group in the histidine-aldol cross-link can, in turn, form a Schiff base with yet another side chain, e.g., hydroxylysine. By such cross-linking reactions, four side chains and two or more molecules can be covalently bonded together.
It has been shown that purified collagen can be utilized medically in reconstructive and cosmetic surgery for the replacement of bony structures or gaps in bony structures, and for filling out tissues where wrinkles have formed. In such usage, collagen is secured from mammalian sources, e.g., calves, and extraneous proteinaceous material is removed by various dissolution, precipitation and filtration techniques to leave a pure collagenous product. Native collagen has limited clinical usefulness since it may induce antigenic response in the host subject. Such response is generated principally by the non-helical terminal portions of the collagen molecule. These end regions can be cleaved by treatment with a proteolytic enzyme, e.g. pepsin. After digestion with pepsin, the cleaved peptide ends are discarded and only the helical domain which comprises more than 95% of the molecule remains. These molecules are of low antigenicity and they can be used for the purposes noted above without undue antigenic side effects.
Unfortunately, however, the helical collagen domain contains little, if any, of the native covalent cross-links that stabilize native collagen and produce its high tensile strength as well as resistance to degradation by enzymes and resorption by body fluids. Thus, the unmodified collagen products available to the medical profession are soon subject to break-down and resorption in a host subject unless artificial, potentially antigenic cross-links are introduced.
It is therefore desirable to devise a low-antigenic collagen product that will exhibit the tensile properties of native collagen and resist degradation and resorption. One obvious technique for achieving such a desired collagen would be the induction of cross-links between various amino-acid residues occuring in the central portions of the collagen helixes. The nature of such cross-links have been noted above. However, such cross-links normally occur in those portions of the collagen that are cleaved off to yield a low-antigenic product.
Some reports have noted the induction of cross-links in the low-antigenic helical collagen by employing glutaraldehyde as an intermediate moiety in the production of cross-links between lysine residues. However, the introduction of glutaraldehyde into the collagen cross-links introduces a new antigenic determinant, and may alter certain physical properties of cross-linked fibrils in undesirable ways.
It is of considerable interest, therefore, to increase the resistance of low-antigenic collagen to degradation when emplaced within body tissues. Such improvement could be provided by inducing native-type cross-linking into the helical low antigenic domain of the collagen molecule.